Confocal laser scanning microscope and a method for examining a sample

ABSTRACT

A confocal laser scanning microscope for examining a sample has a light source, which generates an illumination light beam, and a scanning unit which deflects the illumination light beam such that it optically scans the sample. A main beam splitter separates the illumination light beam from detection light emerging from the sample. The detection light separated from the illumination light beam passes at least partially through a detection pinhole diaphragm. At least two detector units detect the detection light passing through the detection pinhole diaphragm. An optical element is arranged in the beam direction between the detection pinhole diaphragm and the detector units and splits the detection light into at least two beam bundles and spectrally decomposes it within the beam bundles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application under 35 U.S.C.§371 of International Application No. PCT/EP2011/070623, filed on Nov.22, 2011, and claims benefit to German Patent Application No. DE 10 2010060 747.9, filed on Nov. 23, 2010. The international application waspublished in German on May 31, 2012, as WO 2012/069443 A1 under PCTArticle 21(2).

FIELD

The invention relates to a confocal laser scanning microscope and to amethod for investigating a sample.

BACKGROUND

A confocal laser scanning microscope is suitable for investigating amicroscopic sample. For this purpose, fluorescent markers that formconnections to structures of the sample, or to elements participating inprocesses in the sample, are introduced into the sample. The fluorescentmarkers can be activated with the aid of excitation light in such a waythat they are excitable to fluoresce and/or are excited to fluoresce,with the result that the structures and/or processes in the sample aremade visible. Fluorescent light proceeding from the sample, which inthis connection can also be referred to as “detected light,” isseparated from the illumination light and directed via a detectionaperture onto a detector unit.

A scanning unit causes the illumination light beam to optically scan thesample. The detected light is detected as a function of positions of thescanning unit, so that the region of the sample from which the detectedlight is currently deriving is known at every point in time duringdetection, so that an image of the sample can subsequently be created onthe basis of the acquired data.

The wavelength regions of the fluorescent light depend on thefluorescent markers. In other words, different fluorescent markers lightup in different colors when they are excited to fluoresce. It is knownto investigate the individual fluorescent markers, and the structures ofthe sample and/or processes in the sample connected to them,independently of one another by illuminating the sample exclusively withillumination light from a predetermined wavelength region, so that onlya specific type of fluorescent markers is excited to fluoresce; or thedetected light can be filtered with the aid of a color filter in such away that only detected light of one or a few fluorescent makers arrivesat the detector unit.

DE 43 30 347 C2 discloses an apparatus for selecting and detecting atleast two spectral regions of a light beam, in which apparatus a lightbeam is spectrally divided. The divided light beam strikes a mirroraperture that allows part of the light to pass through to a firstdetector unit and reflects the remainder of the light to a seconddetector unit.

SUMMARY

An aspect of the present invention is to provide a confocal laserscanning microscope and a method for investigating a sample that make itpossible, in simple fashion, to investigate different spectral regionsof the detected light.

In an embodiment, the present invention provides a confocal laserscanning microscope for investigating a sample. The microscope includes:a light source configured to generate an illumination light beam; ascanning unit configured to deflect the illumination light beam in sucha way that the illumination light beam optically scans the sample; amain beam splitter configured to separate the illumination light beamfrom detected light proceeding from the sample; a detection apertureconfigured to allow the detected light separated from the illuminationlight beam to pass through the detection aperture, at least in part; atleast two detector units configured to detect the detected light passingthrough the detection aperture; and an optical element disposed betweenthe detection aperture and the detector units in the beam direction,wherein the optical element is configured to separate the detected lightinto at least two beam bundles and spectrally divide the detected lightwithin the beam bundles.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described in even greater detail belowbased on the exemplary figures. The invention is not limited to theexemplary embodiments. All features described and/or illustrated hereincan be used alone or combined in different combinations in embodimentsof the invention. The features and advantages of various embodiments ofthe present invention will become apparent by reading the followingdetailed description with reference to the attached drawings whichillustrate the following:

FIG. 1 shows a confocal laser scanning microscope,

FIG. 2 shows a first embodiment of a detector apparatus of the laserscanning microscope,

FIG. 3 shows a second embodiment of the detector apparatus,

FIG. 4 shows a third embodiment of the detector apparatus, and

FIG. 5 shows a fourth embodiment of the detector apparatus.

DETAILED DESCRIPTION

According to a first aspect, the invention relates to a confocal laserscanning microscope for investigating a sample. The laser scanningmicroscope has a light source that generates an illumination light beam.A scanning unit deflects the illumination light beam in such a way thatit optically scans the sample. A main beam splitter separates theillumination light beam from detected light proceeding from the sample.The detected light separated from the illumination light passes througha detection aperture. A detector apparatus detects the detected lightpassing through the detection aperture.

An aspect of the invention is notable for the fact that an opticalelement is arranged between the detection aperture and the detectorapparatus in the beam direction, and separates the detected light intoat least two beam bundles and spectrally divides it within the beambundles.

The arrangement of the optical element behind the detection aperture inthe detected beam direction allows detection of two of more wavelengthregions of the detected light in the form of two or more beam bundles.This allows the wavelength regions to be detected to be particularlyprecisely separated from one another, determined, and/or detected. Thewavelengths of the beam bundles, and thus the spectral regions of thedetected light that are to be detected, can be unrestrictedly chosenand/or selected automatically. Further advantages result from thepossibility of a particularly robust construction of the laser scanningmicroscope, and from good transmittance values. The laser scanningmicroscope configured in this fashion is moreover also suitable forfluorescence live time microscopy (FLIM) applications, for fluorescencecorrelation spectroscopy (FCS) applications, and for fluorescenceresonance energy transfer (FRET) applications.

In an embodiment, the optical element comprises at least two differentsurfaces, onto which one of the beam bundles from the optical elementrespectively arrives. This can contribute, in simple fashion, todividing the detected light into different beam bundles. The fact thatthe surfaces are “different” means in this context that the surfaces areseparated from one another, for example, by an edge or an inflection.

According to a preferred embodiment, the optical element encompasses aprism arrangement. The prism arrangement can comprise two, three, ormore different prisms. For example, the prism arrangement can compriseone prism for each beam bundle.

An embodiment provides that detected light already dispersed into beambundles is spectrally limited by the fact that the wavelength regions ofthe individual beam bundles are limited. This is accomplished preferablywith the aid of spectrally limiting elements that encompass, forexample, apertures and/or light-guiding fibers. It is particularlyadvantageous in this context if a portion of one of the beam bundlesthat corresponds to the spectrally limited wavelength region is variablein terms of its wavelengths. In other words, the intention is to createthe possibility of variably selecting smaller portions of the detectedlight within the beam bundles and detecting them in targeted fashion.The variability of the portions in terms of their wavelengths can beensured, for example, by a displaceability of the spectrally limitingelement, by the provision of movable mirrors, and/or by rotation ordisplacement of the optical element.

According to a second aspect, the invention relates to a method forinvestigating the sample, in which method the illumination light beam isgenerated and is deflected so that it optically scans the sample. Theillumination light beam is separated from the detected light proceedingfrom the sample. A cross section of the detected light is limited withthe aid of a detection aperture. The limited detected light is detectedwith the aid of detector units. Between the detection aperture and thedetector units in the beam direction, the detected light is separatedinto at least two beam bundles and is spectrally divided within the beambundles.

Elements of identical design or function are labeled with the samereference characters throughout the Figures.

FIG. 1 shows a confocal laser scanning microscope 20. Laser scanningmicroscope 20 comprises a light source 22 that generates an illuminationlight beam 24. Light source 22 encompasses at least one laser thatgenerates light of a specific wavelength, of a small wavelength region,or of a large wavelength region. Light source 22 can, for example,encompass a white light laser that generates broad-band laser light(also referred to as “white light”). Alternatively thereto, two or morelasers can also be provided. Laser scanning microscope 20 is suitablefor many applications in the sector of fluorescence microscopy, and inparticular for the detection of fluorescent light. Laser scanningmicroscope 20 is particularly suitable for separate detection offluorescent light of different fluorescent markers. Laser scanningmicroscope 20 is further suitable for implementing FLIM, FCS, and FRETapplications.

Illumination light beam 24 emerges from laser light source 22 and isdirected via a deflection mirror 26 and a filter 28 onto a first lens30. After passing through first lens 30, illumination light beam 24passes through an illumination aperture 32 and strikes a main beamsplitter 34. Main beam splitter 34 directs illumination light beam 24through a second lens 36 onto a scanning unit 38. Scanning unit 38preferably comprises one or more mirrors, which are coupled topositioning elements in such a way that they are displaceable, forexample in a motion direction 40, in reaction to a control signal.Scanning unit 38 directs illumination light beam 24, through a thirdlens 42 and a fourth lens 44 that form an objective, onto a sample 46that is optically scanned with the aid of illumination light beam 24 asa result of the deflection of illumination light beam 24 by scanningunit 38.

Detected light 48 proceeding from sample 46 travels through third andfourth lens 42, 44, through scanning unit 38, and through second lens 36to main beam splitter 34, which allows detected light 48 to pass througha detection aperture 50 to a detector apparatus 60. Detected light 48 ispreferably fluorescent light. Alternatively thereto, however, detectedlight 48 can also be light reflected from sample 46 or, in the case oftransmitted-light illumination, can also encompass transmitted light.Detected light 48 is detected as a function of positions of scanningunit 38, so that the region of sample 46 from which detected light 48 iscurrently deriving is known at every point in time during detection;this subsequently allows an image of sample 46 to be created.

FIG. 2 shows a first embodiment of detector apparatus 60. Detectorapparatus 60 encompasses an optical element 62 that is preferablyembodied as a prism arrangement. Optical element 62 encompasses a firstprism 64, a second prism 66, and a third prism 68. Optical element 62divides detected light 48 into multiple beam bundles 70, 72, 74. Inparticular, a first beam bundle 70 emerges from a first surface 71 offirst prism 64. A second beam bundle 72 emerges from a second surface 73of second prism 66. A third beam bundle 74 emerges from a third surface75 of third prism 68. First beam bundle 70 encompasses light havingwavelengths of a first wavelength region 80, detected light 48 of secondbeam bundle 72 encompasses detected light 48 of a second wavelengthregion 82, and detected light 48 of third beam bundle 74 encompassesdetected light 48 of a third wavelength region 84. Optical element 62can be embodied, for example, in accordance with a prism arrangementshown in U.S. Pat. No. 4,084,180 A1.

A first spectral segment 92 is cut out of first beam bundle 70 with theaid of a first positionable aperture 86. A second spectral segment 94 ofdetected light 48 is cut out of second beam bundle 72 with the aid of asecond positionable aperture 88. A third spectral segment 96 of detectedlight 48 is cut out of third beam bundle 74 with the aid of a thirdpositionable aperture 90. Segments 92, 94, 96 can also be referred to as“bandwidth segments” of the corresponding beam bundles 70, 72, 74.Segments 92, 94, 96 thus encompass light of small wavelength segmentsthat are cut out of the corresponding wavelength regions 80, 82, 84 ofbeam bundles 70, 72, 74, where beam bundles 70, 72, 74 correspond to thespectrally split detected light 48. In other words, a coarse separationof detected light 48 into beam bundles 70, 72, 74 is accomplished withthe aid of the prism arrangement, and a particularly fine and precisedivision of detected light 48 into segments 92, 94, 96 is accomplishedwithin beam bundles 70, 72, 74. A shifting of positionable apertures 86,88, 90 in corresponding aperture positioning directions 98 allows thewavelengths of segments 92, 94, 96 to be varied. Variation of thewavelengths of segments 92, 94, 96 allows adaptation of detected light48 that is to be detected to different fluorescence maxima of thefluorescent makers used in sample 46.

The remaining detected light 48 in the form of segments 92, 94, 96 isdirected via focusing lenses 100 onto corresponding detector units 102,104, 106, in particular onto a first detector unit 102 that isassociated with first beam bundle 70, onto a second detector unit 104that is associated with second beam bundle 72, and onto a third detectorunit 106 that is associated with third beam bundle 74. Detector units102, 104, 106 encompass, for example, PMTs, APDs, or photodiodes thatconvert the detected light 48 into electrical signals and make itavailable to a control unit (not depicted).

FIG. 3 shows a second embodiment of detector apparatus 60. The secondembodiment corresponds to the first embodiment shown in FIG. 1 in termsof optical element 62 and the division of detected light 48 into beambundles 70, 72, 74, and in terms of detector units 102, 104, and 106associated with beam bundles 70, 72, 74. In contrast to the firstexemplifying embodiment, however, beam bundles 70, 72, 74 are spectrallylimited by the fact that light guides 110, 112, 114 are provided insteadof positionable apertures 86, 88, 90. Light-guiding fibers, for exampleglass fibers, can be used as light guides 110, 112, 114. In particular,first beam bundle 70 is coupled into a first light guide 110, secondbeam bundle 72 into a second light guide 112, and third beam bundle 74into a third light guide 114. A cross section of light guides 110, 112,114 is smaller than the cross section of the corresponding beam bundles70, 72, 74, which brings about the spectral limiting of detected light48 of beam bundles 70, 72, 74. Segments 92, 94, 96 of detected light 48that emerge from light guides 110, 112, 114 strike the correspondingdetector units 102, 104, 106. Segments 92, 94, 96, and in particular thewavelengths that these segments 92, 94, 96 encompass, can be varied bymoving light guides 110, 112, 114 along light guide positioningdirections 116. Light guides 110, 112, 114 are coupled for this purposeto corresponding positioning apparatuses.

FIG. 4 shows a third exemplifying embodiment of the detector apparatus,which corresponds to the second exemplifying embodiment according toFIG. 3 in terms of the shape of prism arrangement 62 and the provisionof light guides 110, 112, 114. For the sake of clarity, depiction ofdetector units 102, 104, 106 and of segments 92, 94, 96 in FIG. 4 hasbeen omitted. In contrast to the second exemplifying embodiment ofdetector apparatus 60, in the third exemplifying embodiment light guides110, 112, 114 are not arranged movably, but instead the prismarrangement is arranged rotatably. In particular, optical element 62, inparticular the prism arrangement, can be rotated so that its side facingtoward detected light beam 48 is parallel to a reference line 118 andthen, after rotation of the prism arrangement, encloses an angle α or −αwith reference line 118. Rotation of the prism arrangement causes beambundles 70, 72, 74 to be emitted in different directions. Because of thespectral division of beam bundles 70, 72, 74, different wavelengthregions of beam bundles 70, 72, 74 enter light guides 110, 112, 114 as afunction of the rotation angle, with the result that the wavelengths ofsegments 92, 94, 96 are varied.

FIG. 5 shows a fourth exemplifying embodiment of detector apparatus 60that corresponds substantially to the exemplifying embodiment accordingto FIG. 4, i.e. to the third exemplifying embodiment. Depiction ofsegments 92, 94, 96 and of detector units 102, 104, 106 has been omittedin FIG. 5. In contrast to the rotatability of the prism arrangementaccording to the fourth exemplifying embodiment, the prism arrangementis fixed. A first mirror 120, a second mirror 122, and a third mirror124 are, however, respectively arranged between the prism arrangementand light guides 110, 112, 114 in the beam direction of beam bundles 70,72, 74. Mirrors 120, 122, 124 are movable along mirror positioningdirections 126 and/or alternatively thereto are arranged rotatably, sothat different spectral regions of beam bundles 70, 72, 74 can becoupled into light guides 110, 112, 114 as a function of positions ofmirrors 120, 122, 124; this has an effect on the wavelengths of segments92, 94, 96 In addition, a rotation of mirrors 120, 122, 124 brings abouta stretching or compression of the light spectrum of segments 92, 94,96, with the result that a bandwidth of the light to be detected can beadjusted.

The exemplifying embodiments of detector apparatus 60 that are shownmake possible a spectrally separated detection of detected light 48, inthe form of beam bundles 70, 72, 74, behind detection aperture 50. Thisoffers a user the possibility of separating and detecting even morefinely, in the form of segments 92, 94, 96, those wavelength regions ofdetected light 48 which are to be detected; in an embodiment that is notshown, further separation in the form of segments 92, 94, 96 can also beomitted, and the entire beam bundles 70, 72, 74 can be detected. Inaddition, alternatively thereto, the separation of segments 92, 94, 96can be accomplished by restricting the sensitive sensor areas ofdetector units 102, 104, 106. In particular, the sensitive sensor areascan be smaller than the cross sections of beam bundles 70, 72, 74, sothat only segments 92, 94, 96 of beam bundles 70, 72, 74 are detected.If this is combined with the rotatable prism arrangement, thewavelengths of segments 92, 94, 96 can then be varied in particularlysimple fashion. The exemplifying embodiments shown can moreover becombined with one another.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive. Itwill be understood that changes and modifications may be made by thoseof ordinary skill within the scope of the following claims. Inparticular, the present invention covers further embodiments with anycombination of features from different embodiments described above andbelow.

The terms used in the attached claims should be construed to have thebroadest reasonable interpretation consistent with the foregoingdescription. For example, the use of the article “a” or “the” inintroducing an element should not be interpreted as being exclusive of aplurality of elements. Likewise, the recitation of “or” should beinterpreted as being inclusive, such that the recitation of “A or B” isnot exclusive of “A and B.” Further, the recitation of “at least one ofA, B, and C” should be interpreted as one or more of a group of elementsconsisting of A, B, and C, and should not be interpreted as requiring atleast one of each of the listed elements A, B, and C, regardless ofwhether A, B, and C are related as categories or otherwise.

PARTS LIST

20 Laser scanning microscope

22 Light source

24 Illumination light beam

26 Deflection mirror

28 Filter

30 First lens

32 Illumination aperture

34 Main beam splitter

36 Second lens

38 Scanning unit

40 Motion direction

42 Third lens

44 Fourth lens

46 Sample

48 Detected light

50 Detection aperture

60 Detector apparatus

62 Optical element

64 First prism

66 Second prism

68 Third prism

70 First beam bundle

71 First surface

72 Second beam bundle

73 Second surface

74 Third beam bundle

75 Third surface

80 First wavelength region

82 Second wavelength region

84 Third wavelength region

86 First positionable aperture

88 Second positionable aperture

90 Third positionable aperture

92 First segment

94 Second segment

96 Third segment

98 Aperture positioning direction

100 Focusing lens

102 First detector unit

104 Second detector unit

106 Third detector unit

110 First light guide

112 Second light guide

114 Third light guide

116 Light guide positioning direction

118 Reference line

120 First detector mirror

122 Second detector mirror

124 Third detector mirror

126 Mirror positioning direction

1. A confocal laser scanning microscope for investigating a sample,having the microscope comprising: a light source configured to generatean illumination light beam; a scanning unit configured to deflect theillumination light beam in such a way that the illumination HAL beamoptically scans the sample; a main beam splitter configured to separatethe illumination light beam from detected light proceeding from thesample; a detection aperture configured to allow the detected lightseparated from the illumination light beam to pass through the detectionaperture, at least in part; at least two detector units, configured todetect the detected light passing through the detection aperture; and anoptical element disposed between the detection aperture and the detectorunits in the beam direction, wherein the optical element is configuredto separate the detected light into at least two beam bundles andspectrally divide the detected light within the beam bundles.
 2. Themicroscope of claim 1, wherein the optical element comprises at leasttwo different surfaces, onto which one of the beam bundles respectivelyarrive.
 3. The microscope of claim 1, wherein the optical elementcomprises a prism arrangement.
 4. The microscope of claim 1, furthercomprising: a spectrally limiting element configured to spectrally limitat least one wavelength region of the beam bundles, corresponding to aspectrally limited wavelength region, prior to detection.
 5. Themicroscope of claim 4, wherein the spectrally limiting element comprisesan aperture.
 6. The microscope of claim 4, wherein a portion of thedetected light that corresponds to the spectrally limited wavelengthregion is variable its terms of its wavelengths.
 7. The microscope ofclaim 6, wherein the portion is variable in terms of its wavelengths bythe spectrally limiting element being displaceably disposed.
 8. Themicroscope of claim 6, further comprising: a detector mirror configuredto direct the beam bundle onto the spectrally limiting element, whereinthe detector mirror is displaceably, rotatably, or dispaceably androtatably so as vary the wavelengths of the portion of the spectrallylimited detected light.
 9. The microscope of claim 6, wherein theoptical element is disposed rotatably, displaceably, or rotatably anddisplaceably, so as vary the wavelengths of the portion.
 10. Themicroscope of claim 1, wherein the optical element is configured toseparate the detected light into at least three or more beam bundles,and wherein the optical element is configured to spectrally divide thelight within the beam bundles.
 11. A method for investigating a sample,the method comprising: generating an illumination light beam; deflectingthe illumination light beam so that the illumination light beamoptically scans the sample; separating the illumination light beam froma detected light proceeding from the sample; limiting a cross section ofthe detected light using detection aperture so as to obtain a limiteddetected light; detecting the limited detected light using one or moredetector units; and between the detection aperture and the detectorunits in a beam direction, separating the detected light into at leasttwo beam bundles and spectrally dividing the detected light within thebeam bundles.
 12. The microscope of claim 4, wherein the spectrallylimiting element comprises a light-guiding fiber.
 13. The microscope ofclaim 4, wherein the spectrally limiting element comprises an apertureand a light-guiding fiber.
 14. The microscope of claim 8, wherein thedetector mirror is arranged displaceably
 15. The microscope of claim 8,wherein the detector mirror is arranged rotatably.
 16. The microscope ofclaim 8, wherein the detector mirror is arranged dispaceably androtatably.
 17. The microscope of claim 9, wherein the optical element isarranged rotatably.
 18. The microscope of claim 9, wherein the opticalelement is arranged displaceably.
 19. The microscope of claim 9, whereinthe optical element is arranged rotatably and displaceably.